No abstract

Light energy for photosynthesis is collected by the antenna system, creating an excited state which migrates energetically 'downhill'. To achieve efficient migration of energy the antenna is populated with a series of pigments absorbing at progressively redshifted wavelengths. This variety in absorbing species in vivo has been created in a biosynthetically economical fashion by modulating the absorbance behaviour of one kind of (bacterio)chlorophyll molecule. This modulation is poorly understood but has been ascribed to pigment-pigment and pigment-protein interactions. We have examined the relationship between aromatic residues in antenna polypeptides and pigment absorption, by studying the effects of site-directed mutagenesis on a bacterial antenna complex. A clear correlation was observed between the absorbance of bacteriochlorophyll a and the presence of two tyrosine residues, alpha Tyr44 and alpha Tyr45, in the alpha subunit of the peripheral light-harvesting complex of Rhodobacter sphaeroides, a purple photosynthetic bacterium that provides a well characterized system for site-specific mutagenesis. By constructing single (alpha Tyr44, alpha Tyr45----PheTyr) and then double (alpha Tyr44, alpha Tyr45----PheLeu) site-specific mutants, the absorbance of bacteriochlorophyll was blueshifted by 11 and 24 nm at 77 K, respectively. The results suggest that there is a close approach of tyrosine residues to bacteriochlorophyll, and that this proximity may promote redshifts in vivo.

A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 °C. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micropatterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nanopatterned PCysMA brushes were obtained using interference lithography, and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX þ and PufX À strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX À cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1b. This arrangement, coupled with a flexible ring, would give the RC Q B site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc 1 complex.

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

No abstract

Förster’s theory of resonant energy transfer underlies a fundamental process in nature, namely the harvesting of sunlight by photosynthetic life forms. The theoretical framework developed by Förster and others describes how electronic excitation migrates in the photosynthetic apparatus of plants, algae, and bacteria from light absorbing pigments to so-called reaction centers where light energy is utilized for the eventual conversion into chemical energy. The demand for highest possible efficiency of light harvesting appears to have shaped the evolution of photosynthetic species from bacteria to plants which, despite a great variation in architecture, display common structural themes founded on the quantum physics of energy transfer as described first by Förster. In this review, Förster’s theory of excitation transfer is summarized including recent extensions, and the relevance of the theory to photosynthetic systems as evolved in purple bacteria, cyanobacteria, and plants is demonstrated. In an Appendix, Förster’s energy transfer formula, as used widely today in many fields of science, is derived.

Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The Photosynthetic organisms synthesize both chlorophyll and heme, the two major tetrapyrroles in nature. The biosynthetic pathways of these two porphyrins utilize a number of intermediates in common and the first step unique to chlorophyll production is the insertion of Mg into protoporphyrin IX (see Fig. 1). The enzyme catalyzing this insertion is known as magnesium-protoporphyrin IX chelatase and it lies at the branch point of the heme and the bacteriochlorophyll/chlorophyll biosynthetic pathways. Despite the importance of (bacterio)chlorophyll biosynthesis, there is relatively little known about the detailed enzymology and protein chemistry of this pathway, and in the case of Mg chelatase biochemical analyses have been confined to assays using intact cells of the photosynthetic bacterium Rhodobactersphaeroides (1, 2), isolated plastids (3-6), and broken plastid systems (7)(8)(9)(10)(11)(12). In these systems, ATP is absolutely required for magnesium chelatase activity (4). Furthermore, it has been demonstrated with extracts of pea (Pisum sativum) chloroplasts that two components, one soluble and the other with membrane affinity, participate in the enzymatic reaction and that there is an ATP requirement for the activation of these two components (10). Recently, the analysis of this pathway in photosynthetic bacteria has provided a way forward (for a review, see ref. 13). This approach benefits from the availability of the genes for bacteriochlorophyll biosynthesis in Rhodobacter capsulatus and R. sphaeroides, which are clustered on a small region of the genome, -45 kb long (14-17). The gene assignments have been based on the results of insertional mutagenesis, which have been correlated with the accumulation of biosynthetic intermediates, or by the measurement of enzymatic activities (15,(17)(18)(19). Positive identification of function has been lacking, but two recent publications describe the overexpression of the bchM gene from both R. sphaeroides and R. capsulatus in Escherichia coli and demonstrate that the extracts of the E. coli transformants can convert Mg-protoporphyrin IX to Mgprotoporphyrin monomethyl ester (20,21). Apart from positively identifying bchM as the gene encoding the Mgprotoporphyrin methyltransferase, this work opens up the possibility of extending this approach to other parts of the pathway. In this paper, we report the expression of the genes bchH, -I, and -D from R. sphaeroides in E. coli: extracts from these transformants, when combined in vitro, are highly active in catalyzing the chelation of Mg by protoporphyrin IX in an ATP-dependent manner. This is an important step forward since apart from identifying the role of three more bch genes-bchH, -I, and -D-it will allow the biochemistry of this reaction to be studied in deta...